Fig. 4: Precise regulation of the CRISPR-Cas12b system by modification of the universal DR in the currently developed split sgRNA.
From: Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b
a Schematic representation of how glyoxal-caged DR effectively inhibits both the cis- and trans-cleavage activities of the CRISPR-Cas12b system in the tracrRNA+DR+Spacer configuration. This function can be precisely restored through temperature- or time-mediated regulation. An alternative approach illustrated here employs a PC linker attached to the DR region, which similarly inhibits the cis- and trans-cleavage activities of CRISPR-Cas12b, with restoration achieved via UV exposure. b Real-time fluorescence data demonstrate the extent of inhibition on trans-cleavage activity by glyoxal-caged DR, showing complete recovery upon heating. A 45-minute exposure to glyoxal-caged DR significantly reduces trans-cleavage activity (0 min). Following a 5-minute incubation at 60 °C, the activity gradually recovers, reaching near-complete restoration by 40 minutes. Data are shown as mean value +/− SD (n = 3, biologically independent samples). c A bar chart illustrates the correlation between incubation time and the recovery rate of cis-cleavage activity for glyoxal-caged DR under 60 °C heating conditions. All the experiments were conducted in triplicate, and error bars represent mean value +/− SD (n = 3). d Real-time fluorescence data suggest the inhibitory effect of an 8 nt random DNA sequence attached to the DR region on Cas12b’s trans-cleavage activity, with restoration observed after 15 seconds of UV light exposure. Importantly, this 15-second UV exposure does not adversely affect the system, and the presence of 8 nt random DNA (DNA1, DNA2) does not disrupt its function. Data are shown as mean value +/− SD (n = 3, biologically independent samples). e Gel electrophoresis results provided supporting evidence for the effective inhibition of CRISPR-Cas12b’s cis-cleavage activity by DR-PC-DNA, with activity recovery observed following 15 seconds of UV light exposure. The experiment was performed three times. f Real-time fluorescence data illustrating the detection of varying copy numbers of EBV using the developed one-pot RPA-CRISPR-Cas12b method. Following a 20-minute RPA reaction, Cas12b activity is activated via 15 seconds of UV irradiation (365 nm, 35 W), resulting in the release of fluorescence signals. Data are shown as mean value +/− SD (n = 3, biologically independent samples). g A heat map comparing results from traditional qPCR methods with those from the developed split sgRNA-assisted one-pot RPA-CRISPR-Cas12b methods applied to different clinical plasma samples from EBV-infected individuals. Error bars represent standard deviation (n = 3). Data are shown as mean ± standard deviation, based on three technical replicates. Source data are provided as a Source Data file. This Fig. 4a is adapted from Wang, J., Zhang, W., Li, W., Xie, Q., Zang, Z. and Liu, C. Enhancement of CRISPR-Cas12a system through universal circular RNA design. Cell Rep. Methods 5, 101076 (2025).62, licensed under CC-BY 4.0 (https://creativecommons.org/licenses/by/4.0/).
